This invention relates to nucleic acid molecules and the encoded GTPase activating proteins, and variants thereof, and to the use of these molecules in the characterization, diagnosis, prevention, and treatment of cell signaling, immune, and cell proliferative disorders, particularly colon cancer.
Guanine nucleotide binding proteins (GTP-binding proteins) are present in all eukaryotic cells and function in processes including metabolism, cellular growth, differentiation, signal transduction, cytoskeletal organization, and intracellular vesicle transport and secretion. In higher organisms they are involved in signaling that regulates such processes as the immune response (Aussel et al (1988) J Immunol 140:215-220), apoptosis, differentiation, and cell proliferation including oncogenesis (Dhanasekaran et al. (1998) Oncogene 17:1383-1394).
The superfamily of GTP-binding proteins consists of several families and may be grouped as translational factors, heterotrimeric GTP-binding proteins involved in transmembrane signaling processes (also called G-proteins), proto-oncogene Ras proteins, and other low molecular weight GTP-binding proteins including the products of rab, rap, rho, rac, smg21, smg25, YPT, SEC4, and ARF genes, and tubulins (Kaziro et al. (1991) Ann Rev Biochem 60:349-400).
The low molecular weight (LMW) GTP-binding proteins are a class of small proteins of 21-30 kDa. These proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. In particular, LMW GTP-binding proteins activate cellular proteins by transducing mitogenic signals involved in various cell functions in response to extracellular signals from receptors (Tavitian (1995) C R Seances Soc Biol Fil 189:7-12). During this process, the hydrolysis of GTP acts as an energy source as well as the means of converting the GTP-binding protein from an active (GTP-bound) to and inactive (GDP-bound) form.
The Rho family of LMW GTP-binding proteins ( Rho GTPases) includes RhoA, Rac 1, and Cdc42 which regulate a variety of biological events related to actin-cytoskeletal reorganization and cell proliferation. A key group of regulatory molecules for the Rho GTPases is the Rho GTPase-activating proteins (GAPs). Rho GAPs preferentially recognize the GTP-bound form of a Rho GTPase and stimulate the intrinsic GTPase activity to hydrolyze the bound GTP to GDP. Rho GAPs therefore function as negative regulators or suppressors of Rho GTPase by stimulating the conversion of the Rho GTPase from the active GTP-bound form to the inactive GDP-bound form (Zhang. et al. (1997) J Biol Chem 272:21999-22007).
Rho GAP proteins share an approximately 170-190 amino acid homology region, designated as the Rho GAP domain, that appears to contain the minimum structural domain necessary for GAP activity (Zheng. et al. (1993) J Biol Chem 24629-24634). Rho GAP proteins share 20-24% amino acid identity in this domain, however, certain specific residues are highly conserved. For example, a pair of arginine residues located near the N-terminus of the Rho GAP domain appear to be highly conserved among Rho GAP proteins and are necessary for maximum catalysis (Leonard et al. (1998) J Biol Chem 273:16210-16215). In addition, a proline-rich, SH3 domain-binding site is also found in the N-terminal region of these proteins (Leonard, supra).
The identification of genes associated with cancer and understanding the genetic mechanisms underlying carcinogenesis are critical to the diagnosis, prevention, and treatment of these diseases. In colon cancer particularly, it is known that a combination of activation of oncogenes, inactivation of tumor suppressor genes, and alteration of DNA mismatch repair genes is involved in the progression from normal mucosa to colon cancer (Fearon et al. (1990) Cell 61:759-767; Chung (1995) Gastroenterology 109:1685-1699). Recently a study was conducted which identified a specific region of chromosome 22 associated with colon cancer based on chromosomal (gene?) allelic losses in colon tumor samples relative to normal colon mucosa (Castells et al. (1999) Gastroenterology 117:8331-837). Using microsatellite markers from chromosome 22, a minimal region of allelic deletion was identified between markers D22S1171 and D22S928 covering an interval of 0.57 cM and corresponding to the cytogenetic location 22q13.33. Various genes on chromosome 22 have been proposed as candidate tumor-suppressor genes associated with colorectal carcinogenesis, however, no mutations have been found in any of these genes (Castells et al. supra).
The discovery of nucleic acid sequences encoding GTPase-activating proteins and variants thereof provides new compositions that are useful in the characterization, diagnosis, prevention, and treatment of cell proliferative disorders, including cancer and, in particular, colon cancer.
The invention is based on the discovery of nucleic acid molecules encoding GTPase-activating protein referred to collectively as xe2x80x9cGTPAPxe2x80x9d and individually as xe2x80x9cGTPAP1 and GTPAP2xe2x80x9d and variants thereof, which satisfies a need in the art by providing compositions useful in the characterization, diagnosis, prevention, and treatment of conditions such as cell signaling, immune, and cell proliferative disorders, particularly colon cancer.
The invention provides isolated and purified human and rat nucleic acid molecules comprising SEQ ID NOs:1-29, and fragments thereof, encoding the mammalian protein comprising the amino acid sequence of SEQ ID NO:30, or portions thereof, a biologically active portion of SEQ ID NO:30, an immunologically active portion of SEQ ID NO:30, and a variant of SEQ ID NO:30 comprising SEQ ID NO:31.
The invention further provides a probe that hybridizes to the mammalian nucleic acid molecules or fragments thereof. The invention also provides isolated and purified nucleic acid molecules that are complementary to the nucleic acid molecules of SEQ ID NOs:1-29. In one aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention further provides a method for detecting a nucleic acid molecule in a sample, the method comprising the steps of hybridizing a probe to at least one nucleic acid molecule of a sample, forming a hybridization complex; and detecting the hybridization complex, wherein the presence of the hybridization complex indicates the presence of the nucleic acid molecule in the sample. In one aspect, the method further comprises amplifying the nucleic acid molecule prior to hybridization. The nucleic acid molecule or a fragment thereof may comprise either an element or a target on a microarray.
The invention also provides a method for using a nucleic acid molecule or a fragment thereof to screen a library of molecules to identify at least one ligand that specifically binds the nucleic acid molecule, the method comprising combining the nucleic acid molecule with a library of molecules under conditions allowing specific binding, and detecting specific binding, thereby identifying a ligand that specifically binds the nucleic acid molecule. Such libraries include DNA and RNA molecules, peptides, PNAs, proteins, and the like. In an analogous method, the nucleic acid molecule or a fragment thereof is used to purify a ligand.
The invention also provides an expression vector containing at least a fragment of the nucleic acid molecule. In another aspect, the expression vector is contained within a host cell. The invention further provides a method for producing a protein, the method comprising the steps of culturing the host cell under conditions for the expression of the protein and recovering the protein from the host cell culture.
The invention also provides substantially purified mammalian GTPAP or a portion thereof. The invention further provides isolated and purified proteins having the amino acid sequence of SEQ ID NO:30, a biologically active portion of SEQ ID NO:30, and an immunologically active portion of SEQ ID NO:30, and a variant of SEQ ID NO:30 comprising SEQ ID NO:31. Additionally, the invention provides a pharmaceutical composition comprising a substantially purified mammalian GTPAP protein or a portion thereof in conjunction with a pharmaceutical carrier.
The invention also provides a method for treating a disease or condition associated with altered expression of GTPAP, comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified mammalian GTPAP protein or a portion thereof in conjunction with a pharmaceutical carrier. In one embodiment of the invention, the disease or condition is selected from cell signaling, immune, and cell proliferative disorders, particularly colon cancer. In another embodiment of the invention, the cancer is a colon cancer.
The invention further provides a method for using at least a portion of the mammalian protein to produce antibodies. The invention also provides a method for using a mammalian protein or a portion thereof to screen a library of molecules to identify at least one ligand that specifically binds the protein, the method comprising combining the protein with the library of molecules under conditions allowing specific binding, and detecting specific binding, thereby identifying a ligand that specifically binds the protein. Such libraries include DNA and RNA molecules, peptides, agonists, antagonists, antibodies, immunoglobulins, drug compounds, pharmaceutical agents, and other ligands. In one aspect, the ligand identified using the method modulates the activity of the mammalian protein. In an analogous method, the protein or a portion thereof is used to purify a ligand. The method involves combining the mammalian protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and separating the protein from the ligand to obtain purified ligand.
The invention further provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the natural mammalian nucleic acid molecule. The invention also provides a method for using the mammalian nucleic acid molecule to produce a mammalian model system, the method comprising constructing a vector containing the mammalian nucleic acid molecule; introducing the vector into a totipotent mammalian embryonic stem cell; selecting an embryonic stem cell with the vector integrated into genomic DNA; microinjecting the selected cell into a mammalian blastocyst, thereby forming a chimeric blastocyst; transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric mammal containing at least one additional copy of mammalian nucleic acid molecule in its germ line; and breeding the chimeric mammal to generate a homozygous mammalian model system.